Aequorea green fluorescent protein analysis by flow cytometry
- Genentech, Inc., San Francisco, CA (United States); and others
The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types. The longer wavelength peak (470 nm) of GFP`s bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T- GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm. 29 refs., 5 figs.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 456820
- Journal Information:
- Cytometry, Vol. 21, Issue 4; Other Information: PBD: 1 Dec 1995
- Country of Publication:
- United States
- Language:
- English
Similar Records
Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine
Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging
Related Subjects
BASIC STUDIES
44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
PROTEINS
BIOLUMINESCENCE
MUTATIONS
MUTANTS
SENSITIVITY
ABSORPTION SPECTRA
GENE REGULATION
BIOLOGICAL MARKERS
ABSORPTION SPECTROSCOPY
SIGNAL CONDITIONING
EVALUATION
CELL FLOW SYSTEMS
FLUORESCENCE
FLUORESCEIN
LASERS